Gene expression profiles of bovine longissimus muscle as affected by dietary n-3 v. n-6 fatty acid (FA) intervention were analysed by microarray pre-screening of >3000 muscle biology/meat quality-related genes as well as subsequent quantitative RT-PCR gene expression validation of genes encoding lipogenesis-related transcription factors (CCAAT/enhancer-binding protein β, sterol regulatory element-binding transcription factor 1), key-lipogenic enzymes (acetyl-CoA carboxylase α (ACACA), fatty acid synthase (FASN), stearoyl-CoA desaturase (SCD)), lipid storage-associated proteins (adipose differentiation-related protein (ADFP)) and muscle biology-related proteins (cholinergic receptor, nicotinic, α1, farnesyl diphosphate farnesyl transferase 1, sema domain 3C (SEMA3C)). Down-regulation of ACACA (P = 0·00), FASN (P = 0·09) and SCD (P = 0·02) gene expression upon an n-3 FA intervention directly corresponded to reduced SFA, MUFA and total FA concentrations in longissimus muscle, whereas changes in ADFP (P = 0·00) and SEMA3C (P = 0·05) gene expression indicated improved muscle function via enhanced energy metabolism, vasculogenesis, innervation and mediator synthesis. The present study highlights the significance of dietary n-3 FA intervention on muscle development, maintenance and function, which are relevant for meat quality tailoring of bovine tissues and modulating animal production-relevant physiological processes.